Eder Zucconi

MULTIPOTENT STEM CELLS FROM UMBILICAL CORD: CORD IS RICHER THAN BLOOD

The identification of mesenchymal stem cell (MSC) sources that are easily obtainable is of utmost importance. Several studies have shown that MSCs could be isolated from umbilical cord (UC) units. However, the presence of MSCs in umbilical cord blood (UCB) is controversial. A possible explanation for the low efficiency of MSCs from UCB is the use of different culture conditions by independent studies. Here, we compared the efficiency in obtaining MSCs from unrelated paired UCB and UC samples harvested from the same donors. Samples were processed simultaneously, under the same culture conditions. Although MSCs from blood were obtained from only 1 of the 10 samples, we were able to isolate large amounts of multipotent MSCs from all UC samples, which were able to originate different cell lineages. Since the routine procedure in UC banks has been to store the blood and discard other tissues, such as the cord and/or placenta, we believe our results are of immediate clinical value. Furthermore, the possibility of originating different cell lines from the UC of neonates born with genetic defects may provide new cellular research models for understanding human malformations and genetic disorders, as well as the possibility of testing the effects of different therapeutic drugs.

Isolation, characterization, and differentiation potential of canine adipose-derived stem cells.

Adipose tissue may represent a potential source of adult stem cells for tissue engineering applications in veterinary medicine. It can be obtained in large quantities, under local anesthesia, and with minimal discomfort. In this study, canine adipose tissue was obtained by biopsy from subcutaneous adipose tissue or by suction-assisted lipectomy (i.e., liposuction). Adipose tissue was processed to obtain a fibroblast-like population of cells similar to human adipose-derived stem cells (hASCs). These canine adipose-derived stem cells (cASCs) can be maintained in vitro for extended periods with stable population doubling and low levels of senescence. Immunofluorescence and flow cytometry show that the majority of cASCs are of mesodermal or mesenchymal origin. cASCs are able to differentiate in vitro into adipogenic, chondrogenic, myogenic, and osteogenic cells in the presence of lineage-specific induction factors. In conclusion, like human lipoaspirate, canine adipose tissue may also contain multipotent cells and represent an important stem cell source both for veterinary cell therapy as well as preclinical studies.

Human multipotent adipose-derived stem cells restore dystrophin expression of Duchenne skeletal-muscle cells in vitro

Background information. DMD (Duchenne muscular dystrophy) is a devastating X‐linked disorder characterized by progressive muscle degeneration and weakness. The use of cell therapy for the repair of defective muscle is being pursued as a possible treatment for DMD. Mesenchymal stem cells have the potential to differentiate and display a myogenic phenotype in vitro. Since liposuctioned human fat is available in large quantities, it may be an ideal source of stem cells for therapeutic applications. ASCs (adipose‐derived stem cells) are able to restore dystrophin expression in the muscles of mdx (X‐linked muscular dystrophy) mice. However, the outcome when these cells interact with human dystrophic muscle is still unknown.

Mesenchymal Stem Cells Derived From Canine Umbilical Cord Vein—A Novel Source for Cell Therapy Studies

The canine model provides a large animal system to evaluate many treatment modalities using stem cells (SCs). However, only bone marrow (BM) protocols have been widely used in dogs for preclinical approaches. BM donation consists of an invasive procedure and the number and differentiation potential of its mesenchymal stem cells (MSCs) decline with age. More recently, umbilical cord was introduced as an alternative source to BM since it is obtained from a sample that is routinely discarded. Here, we describe the isolation of MSCs from canine umbilical cord vein (cUCV). These cells can be obtained from every cord received and grow successfully in culture. Their multipotent plasticity was demonstrated by their capacity to differentiate in adipocytic, chondrocytic, and osteocytic lineages. Furthermore, our results open possibilities to use cUCV cells in preclinical trials for many well-characterized canine model conditions homologs to human diseases.

Human fallopian tube: a new source of multipotent adult mesenchymal stem cells discarded in surgical procedures.

BackgroundThe possibility of using stem cells for regenerative medicine has opened a new field of investigation. The search for sources to obtain multipotent stem cells from discarded tissues or through non-invasive procedures is of great interest. It has been shown that mesenchymal stem cells (MSCs) obtained from umbilical cords, dental pulp and adipose tissue, which are all biological discards, are able to differentiate into muscle, fat, bone and cartilage cell lineages. The aim of this study was to isolate, expand, characterize and assess the differentiation potential of MSCs from human fallopian tubes (hFTs).MethodsLineages of hFTs were expanded, had their karyotype analyzed, were characterized by flow cytometry and underwent in vitro adipogenic, chondrogenic, osteogenic, and myogenic differentiation.ResultsHere we show for the first time that hFTs, which are discarded after some gynecological procedures, are a rich additional source of MSCs, which we designated as human tube MSCs (htMSCs).ConclusionHuman tube MSCs can be easily isolated, expanded in vitro, present a mesenchymal profile and are able to differentiate into muscle, fat, cartilage and bone in vitro.

SJL Dystrophic Mice Express a Significant Amount of Human Muscle Proteins Following Systemic Delivery of Human Adipose‐Derived Stromal Cells Without Immunosuppression

Limb‐girdle muscular dystrophies (LGMDs) are a heterogeneous group of disorders characterized by progressive degeneration of skeletal muscle caused by the absence of or defective muscular proteins. The murine model for limb‐girdle muscular dystrophy 2B (LGMD2B), the SJL mice, carries a deletion in the dysferlin gene that causes a reduction in the protein levels to 15% of normal. The mice show muscle weakness that begins at 4–6 weeks and is nearly complete by 8 months of age. The possibility of restoring the defective muscle protein and improving muscular performance by cell therapy is a promising approach for the treatment of LGMDs or other forms of progressive muscular dystrophies. Here we have injected human adipose stromal cells (hASCs) into the SJL mice, without immunosuppression, aiming to assess their ability to engraft into recipient dystrophic muscle after systemic delivery; form chimeric human/mouse muscle fibers; express human muscle proteins in the dystrophic host and improve muscular performance. We show for the first time that hASCs are not rejected after systemic injection even without immunosuppression, are able to fuse with the host muscle, express a significant amount of human muscle proteins, and improve motor ability of injected animals. These results may have important applications for future therapy in patients with different forms of muscular dystrophies.

Gene Expression Profile of Mesenchymal Stem Cells from Paired Umbilical Cord Units: Cord is Different from Blood

Mesenchymal stem cells (MSC) are multipotent cells which can be obtained from several adult and fetal tissues including human umbilical cord units. We have recently shown that umbilical cord tissue (UC) is richer in MSC than umbilical cord blood (UCB) but their origin and characteristics in blood as compared to the cord remains unknown. Here we compared, for the first time, the exonic protein-coding and intronic noncoding RNA (ncRNA) expression profiles of MSC from match-paired UC and UCB samples, harvested from the same donors, processed simultaneously and under the same culture conditions. The patterns of intronic ncRNA expression in MSC from UC and UCB paired units were highly similar, indicative of their common donor origin. The respective exonic protein-coding transcript expression profiles, however, were significantly different. Hierarchical clustering based on protein-coding expression similarities grouped MSC according to their tissue location rather than original donor. Genes related to systems development, osteogenesis and immune system were expressed at higher levels in UCB, whereas genes related to cell adhesion, morphogenesis, secretion, angiogenesis and neurogenesis were more expressed in UC cells. These molecular differences verified in tissue-specific MSC gene expression may reflect functional activities influenced by distinct niches and should be considered when developing clinical protocols involving MSC from different sources. In addition, these findings reinforce our previous suggestion on the importance of banking the whole umbilical cord unit for research or future therapeutic use.

New Source of Muscle-Derived Stem Cells with Potential for Alveolar Bone Reconstruction in Cleft Lip and/or Palate Patients

Cleft lip and palate (CLP), one of the most frequent congenital malformations, affects the alveolar bone in the great majority of the cases, and the reconstruction of this defect still represents a challenge in the rehabilitation of these patients. One of the current most promising strategy to achieve this goal is the use of bone marrow stem cells (BMSC); however, isolation of BMSC or iliac bone, which is still the mostly used graft in the surgical repair of these patients, confers site morbidity to the donor. Therefore, in order to identify a new alternative source of stem cells with osteogenic potential without conferring morbidity to the donor, we have used orbicular oris muscle (OOM) fragments, which are regularly discarded during surgery repair (cheiloplasty) of CLP patients. We obtained cells from OOM fragments of four unrelated CLP patients (CLPMDSC) using previously described preplating technique. These cells, through flow cytometry analysis, were mainly positively marked for five mesenchymal stem cell antigens (CD29, CD90, CD105, SH3, and SH4), while negative for hematopoietic cell markers, CD14, CD34, CD45, and CD117, and for endothelial cell marker, CD31. After induction under appropriate cell culture conditions, these cells were capable to undergo chondrogenic, adipogenic, osteogenic, and skeletal muscle cell differentiation, as evidenced by immunohistochemistry. We also demonstrated that these cells together with a collagen membrane lead to bone tissue reconstruction in a critical-size cranial defects previously induced in nonimmunocompromised rats. The presence of human DNA in the new bone was confirmed by PCR with human-specific primers and immunohistochemistry with human nuclei antibodies. In conclusion, we showed that cells from OOM have phenotypic and behavior characteristics similar to other adult stem cells, both in vitro and in vivo. Our findings suggest that these cells represent a promising source of stem cells for alveolar bone grafting treatment, particularly in young CLP patients.

Ringo, a Golden Retriever Muscular Dystrophy (GRMD) dog with absent dystrophin but normal strength

The closest model to human Duchenne Muscular Dystrophy (DMD) is the Golden Retriever Muscular Dystrophy (GRMD) dog, which carries a point mutation in the splice acceptor site in intron 6 of the orthologe X-linked dystrophin gene, leading to the absence of protein in the muscles. These dogs present clinical signs within the first weeks of life involving the limbs as well as masticatory muscles. Diaphragmatic and intercostal muscles impairment leads to progressive respiratory failure. Death occurs from bronchopneumonia and cardiac arrest, usually before 2 years of age. Here, we report the case of Ringo, an exceptional GRMD dog showing an unusually mild course. Currently, at age 4 years and 10 months he is able to run, jump and open doors while standing on his rear paws. He was also able to breed naturally, which apparently has never been reported before. Ringo is descendant of Beth, a GRMD female carrier donated by Dr. Joe Kornegay (University of North Carolina, USA), and all affected descendants carry the same original mutation. The diagnosis in all dogs was established right after birth through DNA genotyping and elevated serum creatine kinase (CK). At birth, Ringo’s serum CK level was increased 10-fold as compared to his three normal sibs. One affected brother had a comparable serum CK while the other had a 20-fold increase. At 15 days serum CK in the affected dogs was 4to 5-fold higher than in normal siblings. Pedigree analysis (Fig. 1) revealed that Ringo had two affected brothers from the same litter. One of them died at 2 weeks of age. The other one is still alive but with a

Mesenchymal stem cells from umbilical cord: do not discard the cord!

On 10th February 2007, English entrepreneur Sir Richard Branson, best known for his Virgin brand of over 360 companies, announced the establishment of a cord blood bank, the Virgin Health Bank, in London. What makes this bank different is a dual public–private approach. A fifth of the cord blood would be stored for private use for the child or a family member and the rest would be donated to the public part of the bank which will be accessible to anyone in the world who needs it, at no cost. Additionally, Branson has pledged to donate his 50% of proceeds from Virgin Health Bank to researchers investigating the potential of cord blood stem cells [1]. The idea for the dual bank emerged when he was visited by a senior director of the National Blood Center, asking for his support in a charitable role, because children were dying through lack of umbilical cord blood. Initially Sir Richard offered 3 million pounds to the National Health Service to help them increase their storage capacity for umbilical cord blood, but the center was not comfortable with accepting funds from private sources. So Sir Richard decided to set up a company to do the job. This is an initiative that should be applauded but we want to draw attention to a very important and urgent aspect. The routine procedure in umbilical cord banks has been to store the blood and discard other tissues, such as the cord and/or placenta, which is a much better source of mesenchymal stem cells than blood [2]. Umbilical cord